The investigator will study membrane protein components of the E. coli secretion machinery. These components include SecD, SecE, SecF and SecY. Recently, Drs. Mizushima and Wickner have obtained evidence that a fifth membrane protein called SecG (Previously called Band1 or p12) is also part of the apparatus. There is evidence that SecY, E and G form a complex and that SecD and SecF do also. The investigator focuses on SecD, E and F, although since these proteins may all interact, studies inevitably will involve analysis of SecY and SecG. Studies on these proteins have even more significance as a result of recent findings of Dr. Rapoport and his coworkers. It appears that the core secretion machinery in the endoplasmic reticular membrane of eukaryotic cells comprises the same set of proteins as in bacteria. In particular, homologues of SecY and SecE, as well as a third membrane protein have been identified. Thus, understanding of the functioning of the membrane components in E. coli should contribute to studies in eukaryotic cells. The first part of the proposal is designed to determine the important regions of the SecE protein and their role in protein secretion. The SecE protein presents a particularly attractive subject for structure function analysis because of its small size. It is a 13.6 kilodalton (kD) membrane protein which is essential for protein secretion, interacts with at least SecY, and has already been extensively analyzed in this laboratory. Dr. Beckwith has identified an amino acid sequence motif located in a cytoplasmic domain of SecE which is conserved across species. In order to determine the important features of this motif, he will carry out extensive mutagenic analysis of it and test mutants for their ability to complement a secE null mutation. He will then proceed to determine the function that is defective in these mutants, thus asking for information on the role of this protein domain. First, he will determine whether the mutations (and a deletion of this region previously obtained) affect or eliminate binding of SecE to SecY. This can be done by co-overexpressing SecY with the altered SecE's. SecE stabilizes SecY against degradation, providing an easy assay for interaction. Co-precipitation of the two proteins provides another assay for interaction. In addition, in collaboration with the laboratory of Dr. W. Wickner at Dartmouth Medical School, the investigator will study the effects of the mutationally altered SecE's on the functioning of the in vitro protein secretion system. Analysis in vitro of such steps as binding of SecY, SecG and SecA, ATP hydrolysis and extent of translocation should indicate at what stage the process has been altered. The second part of the proposal deals with the role of SecD and SecF in protein secretion. In order to determine the step in protein export that is altered, two approaches will be used. First, Dr. Beckwith will characterize suppressors of a secD,F null mutation. (The null can grow, albeit very poorly at 37 degrees). The nature of the suppressor genes may indicate the function they are replacing. Second, he will seek conditional mutations in secD and secF that shut off protein secretion rapidly. These will be used to determine the step in export that is blocked after shift to non-permissive conditions.